The Immunology Team, which is now part of the Lymphocyte Biology Section, Laboratory of Systems Biology, reported previously on a top-down analysis of the immune and non-immune tissue response of mice to various strains of influenza virus. Highly standardized preparations of both mildly pathogenic (Tx91) and highly pathogenic (PR8) viruses were used at varying infectious doses in a single inbred strain of mouse and several hundred highly qualified microarray transcriptional analyses conducted with RNA isolated from infected lung tissues of these mice at varying time points post-inoculation. Modular gene set analysis uncovered marked differences in transcriptional responses with the PR8 vs. Tx91 strains of virus that can be assigned to specific biological processes. Principal component analysis (PCA) revealed that the transcriptional responses to infection results in cell-type specific changes that largely reflect cell recruitment into the lungs and also context (infection-type) specific changes within the cell-specific gene sets. Furthermore, gene sets related to inflammatory responses are the major component associated with lethality, whereas anti-viral gene sets are similar with both the low and high pathogenicity viruses. A positive feedback pathway involving virus-induced chemokine production facilitates recruitment of myeloid cells to the lungs. Together, these data suggested that uninterrupted amplification of myeloid cell recruitment and inflammatory cytokine production plays a key role in pathogenic infections. In support of this model, attenuating but not eliminating myeloid cell recruitment using depleting antibodies rescues mice from early lethality of PR8 infection. Thus, this study uncovered a core feedback circuit involving innate inflammation that drives early lethality in influenza infection and provides new targets for intervention in this disease. We are now extending this work in an effort to develop a robust computational framework for utilizing peripheral blood transcriptomic data to define tissue-specific pathology. To date, this has proven very difficult, in large measure because the multiple testing correction needed when using whole genome transcriptional data and no starting hypothesis can hide small but significant signals within the blood signature. We are testing whether our tissue-based definition of a lethal signature can be used as a hypothesis to reduce the effects of such multiple testing and hence reveal weak signals in blood-based data sets. If successful (that is, if we can predict lethality from blood data early after influenza infection using guidance from tissue data), this may provide a general way to use limited tissue information to generate predictive blood signatures for use in human disease studies. A second major project involves use of the emerging tools of systems biology to investigate the unexplored roles of many NLRs, a family of sensors found in key immune cells that provide a highly effective first line of defense against infection. In one part of this project, we have created constitutively active versions of many NLRs through truncation of the ligand-sensing LRR domain, expressed these activated forms using tet regulated lentiviruses, and conducted extensive transcriptional profiling of the induced cells. These data are being mined for information on functional biologic pathways activated by these molecules, providing clues to the signaling pathways utilized by the NLR to control cell function. In the course of these studies, we made the unexpected observation that less than a two fold overexpression in one particular NLR leads to the same change in gene expression as 20 fold overexpression of the intact or activated, truncated version of the molecule. Interestingly, this molecule is linked to a number of autoimmune diseases in GWAs studies, suggesting the possibility that even subtle differences in gene regulation that change the steady-state level of the molecule on a long term basis can sensitize the host for inflammatory responses. Additional informatic analyses suggested that several miRNAs might play an important role in tightly controlling the expression of this NLR under normal conditions, and these miRNAs are also linked in GWAS studies to autoimmune conditions. We are presently testing whether inactivation of the miRNAs results in sufficient overexpression of the NLR in non-transduced cells to give global inflammatory gene activation and to develop a model that can explain how such a small difference in mean expression results in such dramatic functional effects. We are also pursuing our published evidence that MAVS plays an important role in NLRP3 inflammasome activation to non-crystalline ligands, in addition to its role in RIG-I-dependent viral sensing. In FY15, we have generated and expressed chimeric molecules that contain different domains RIG-I and NLRP3 or of MAVS, in an effort to dissect how RIG-I links via MAVS to type 1 interferon responses whereas NLRP3 links to ACS and IL-1b responses. As part of the larger LSB group effort to better understand TLR signaling in macrophages, we have conducted fine grained time and dose studies at the single cells level and on bulk populations looking at a diverse set of downstream signaling events and effector responses. Among several intriguing observations, most striking is the discovery that contrary to many models with non-hematopoietic cells, we see a graded response to NFkB in the presence of the expected digital responses among MAPK pathways. The MAPK responses require higher concentration of TLR ligand than the NFkB responses and in the dose region in which NFkB is activated but MAPK responses are not, cells show activation of a subset of NFkB-responsive genes but not transcription of inflammatory cytokine genes. Only when both NFkB and MAPK signals are present are inflammatory cytokines and chemokines generated. These data suggest that the macrophage response system has evolved to limit potentially damaging inflammation in the face of minor pulses of PAMP or DAMP signals engaging TLR (the steady-state), but to prepare for anti-pathogen responses under such conditions incase these weak signals are not from commensals or normal tissue turnover but from an incipient infection; if the stimulus continued to increase, as would typically be the case mainly when there is an active pathogenic infection, then the digital nature of the MAPK pathways in the context of priming through NFkB at lower ligand levels gives an immediate robust response. These data also explain observations of M. Karin that chronic NFkB activation does not lead to intestinal cancer development unless a MAPK stimulus is added. Remarkably, both humans and various mouse strains all share nearly identical dose-responses and among random human donors from the Blood Bank, the responses are nearly identical, a very unusual result in comparison to data on normal volunteers in the CHI influenza and other studies. The data from this project are also serving to drive development of new Simmune-based signaling models for the TLR pathway that has already revealed an unexpected feedback loop from p38 to Erk that is central to the dose-control we observe. This set of observation is being extended to see if the GWAS hits involving DUSPs that are key to the Simmune model help explain the basis for human inflammatory disease as a disturbance on this tightly regulated pathway. We are also proceeding with related studies using combinations of TLR inputs to develop and understanding of how macrophages decode the multiple PAMP signals in pathogens. Finally, we played a major role in organizing and executing a large scale study of the human immune response to influenza vaccine as part of the efforts of the NIH Center for Human Immunology.